Figure 3.

Inhibitory effect of single 8-oxoG on the expression of the inducible EGFP gene. (A) Scheme of the tetracycline-regulated (tet-on) EGFP expression vector. Two TetR binding motifs (TetO₂ ×2) were introduced into the 5′-untranslated region without affecting the protein-coding sequence. Tandem Nt.Bpu10I nicking sites were retained and used for incorporation of synthetic oligonucleotides, as above. (B) Verification of the incorporation of 8-oxoG into the tet-on vector. DNA strand scission analysis of constructs produced with unmodified synthetic oligonucleotide (G) or the oligonucleotide containing single 8-oxoG (8oG). (C) Representative fluorescence distribution plots of T-REx™-Hela cells 24 h post-transfection with the expression constructs containing synthetic oligonucleotides with or without 8-oxoG. Cells were incubated in the absence (- tet) or in the presence of tetracycline (+ tet). (D) Relative EGFP expression calculated at the uninduced (- tet) and induced (+ tet) conditions (n = 6, ± SD).